I’ve been trying to simultaneously extract bacterial DNA and fungal DNA from swabs of skin samples, but I seem to be having some form of contamination. This may take a minute to explain, but let me try to walk through everything that I have done so far.
First, for the attached image, the “blank swabs” were sterile swabs that I ran through the entire extraction and PCR process, but as you can see, I am getting double bands even though they shouldn’t have any DNA (they were run at an annealing temp of 55C for 35 cycles). This gel is just for my fungal primers (I blacked out the bottom right because those samples were from something else); however, I do not get this type of contamination when I run bacterial primers on the same samples (they produce nice single bands).
This does not appear to be contamination at the PCR step because my PCR negative control (sterile water) did not amplify (which has consistently been the case on all tests).
Samples 1 and 2 should have had fungal DNA, but they should just have had a single band (like the positive control) not these bizarre double bands. Also, on this gel I was using a gradient PCR (starting at 47C on the left of each sample and going up to 62.7C on the right of each sample). For sample 1, I ran it at both 35 cycles and 30 cycles. Thus, these double bands do not appear to be an artifact of annealing temperature or number of cycles (especially since supposedly sterile swabs produced double bands).
From all of this, my initial thought was that my extraction reagents were contaminated, which is where things get complicated. Prior to the past few gels, I have been testing multiple kits and methods, and I only recently started getting double bands like this. They showed up on a run where I was comparing CTAB and DNeasy, and they showed up for the results from both methods, even though I was obviously using different chemicals for each. Also, I had previously used both of those kits/reagents without getting double bands (the positive control on the attached image is actually from an earlier test). Further, following my first test that produced double bands, I made an entirely new batch of reagents for CTAB, and I’m still getting the double bands. So this problem just suddenly and simultaneously started for two different methods, and it persists even after making new reagents (also, nothing that I am aware of changed between the runs where I wasn’t getting double bands and the first run where I started getting them).
Finally, it is worth mentioning that I have two different sets of fungal primers (ITS3_KYO2/ITS4 from Toju et al. 2012 and LSU-200F/LSU-841-R from Asemaninejad et al. 2016), and both sets of primers give me double bands (or occasionally even triples). Again though, bacterial primers give me a nice single band for the same samples.
I’m at my wits end, so any suggestions would be greatly appreciated. Thanks.
Hi,
Have you try to change your bag of tubes and tips used to extract ?
Maybe, you can extract those two bands from the the agarose gel and sequence them separately to know if and which contaminant you have there...
Yes, I eventually figured it out. It was an irritating combination of contamination and non-specific primers (I would not recommend the SU-200F/LSU-841-R primer pair)
Tanvi, basically, we just went through one component at a time trying to eliminate things and, in my case, the biggest problem seemed to be that the primers we were using were actually picking up cotton DNA from the swabs we were using (even though they were supposed to be DNA free). We did a primer blast for the primers against a cotton genome and they were a very good match. So just switching primers cleared up most of it. Also, cutting back on the number of PCR runs that we were doing helped clear up some other noise in the gels as well.
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